Hypoxia, DNA repair, and gene silencing. Hypoxia is a key feature of solid tumors that confers radiation resistance, stimulates angiogenesis, promotes metastasis, and is linked to poor prognosis. With the support of this grant, we have shown that hypoxia is also a driver of genetic instability via down-regulation of critical DNA repair genes. In the past funding period, we have discovered that hypoxia can also induce durable silencing of the BRCA1 and MLH1 promoters via specific epigenetic factors. In recent preliminary studies, we have further determined that hypoxia can lead to silencing of the pro-apoptotic BIM gene and resistance to the EGFR inhibitor, gefitinib, in lung cancer cells. The broad, long-term goal of this renewal application is to elucidate the impact of hypoxic stress on carcinogenesis and cancer biology, with a focus on DNA repair and gene silencing. In Aim 1, we will dissect the molecular mechanisms by which hypoxic stress drives epigenetic change to cause gene silencing, with a focus on the MLH1 promoter. We will determine promoter elements and regulatory factors that mediate silencing, and we will use a facile selection-based shRNA screen to identify key targets for reversal of this process. We will also assay for the impact of the hypoxic tumor microenvironment on gene silencing during tumor growth in vivo. Next, since heavy metals are known human carcinogens that can induce hypoxia-related pathways, in Aim 2 we will ask whether exposure to heavy metals can also drive gene silencing and/or down-regulate DNA repair. In Aim 3, we will build on novel preliminary results suggesting that growth of lung cancer cells in hypoxia can promote resistance to the epidermal growth factor receptor (EGFR) inhibitor, gefitinib, in conjunction with silencing of the pro-apoptotic factor, BIM. We wil test specific hypotheses regarding the underlying mechanisms, and, guided by Aim 1, we will identify strategies to prevent or reverse this resistance. The proposed work will elucidate key pathways of gene silencing and DNA repair regulation in response to hypoxia (and possibly to carcinogenic heavy metals) that may underlie critical steps in carcinogenesis, genetic instability, tumor progression, and resistance to radiation and other cancer therapies. Identification of strategies to prevent or reverse these pathways may provide the basis for new approaches to cancer prevention and therapy.